Protein stabilization by domain insertion into a thermophilic protein

ABSTRACT

A strategy to improve protein stability by domain insertion. TEM 1 beta-lactamase (BLA) and exo-inulinase, as model target enzymes, are inserted into a hyperthermophilic maltose binding protein from  Pyrococcus furiosus  (PfMBP). Unlike conventional protein stabilization methods that employ mutations and recombinations, the inventive approach does not require any modification on a target protein except for its connection with a hyperthermophilic protein scaffold. For that reason, target protein substrate specificity was largely maintained, which is often modified through conventional protein stabilization methods. The insertion was achieved through gene fusion by recombinant DNA techniques

STATEMENT OF RELATED APPLICATIONS

This patent application claims the benefit of U.S. provisional patentapplication No. 61/158,124 having a filing date of 6 Mar. 2006, which isincorporated herein in its entirety by this reference.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates generally to the field of stabilizingproteins, and more specifically to the field of stabilizing proteinswithout any modification of their primary sequence. The presentinvention further relates to stabilizing proteins by employing domaininsertion of a target protein into a thermophilic scaffold protein.

2. Prior Art

High specificity and selectivity of a protein as a catalyst are of greatimportance in the (bio)chemical industry as these properties can reducethe number of reaction steps in synthesis and simplify productpurification. For example, lipase has been employed for producing novelpolymers that would otherwise be difficult to make by conventionalchemical polymerization. However, despite a number of advantages overconventional chemical reactions, the progress of enzymatic reactions hasbeen limited due to the insufficient stability of enzymes under commonreaction conditions, such as the presence of organic solvents as well ashigh pressures and temperatures. Under these conditions, proteins unfoldand lose activity significantly. In fact, limited stability is a commonproblem associated with most proteins.

Improvements in stability have been accomplished through rational,combinatorial and data-driven design. A large body of data hasdemonstrated that protein stabilization can be achieved by rational orcombinatorial design or a combination of both. The rational designrequires a knowledge of protein 3D structures and/or an understanding offorces and interactions affecting protein stability. Successful attemptshave been reported in the rational design of highly stable proteins.Some rational protein stabilization strategies include “entropicstabilization” through rigidification by mutations, introduction ofdisulfide bridges, salt bridges, and clusters of aromatic-aromaticinteractions, and engineering of subunit interfaces of multimericproteins. Structural studies of extremophilic organisms and theirproteins have provided significant insight into the moleculardeterminants of stabilization. Mesophilic proteins have been engineeredto become highly stable through mutations found in correspondingthermophilic proteins. Comparative studies on a large number ofnaturally found or engineered stable proteins have revealed theexistence of different ways of enhancing protein stability throughmutations and recombinations. The combinatorial design requiresconstruction of a diverse library and its screening to isolate variantswith desired properties. A considerable amount of proteins with highstability have been identified using combinatorial design. Recently,data-driven design, where the library size is reduced by pinpointingspecific residues to target based on structure and sequence information,has led to isolation of stable proteins.

Improvements in stability also have been accomplished by the addition ofmolecular chaperones and ligands. Molecular chaperones have been usednot only for improving folding of a protein in vivo but also stabilityof a protein in vitro. Exposed hydrophobic surfaces, which should beburied in otherwise native protein structures, are the main targets ofmolecular chaperones. Addition of GroES, GroEL and ATP in vitroincreased kinetic stability of alcohol dehydrogenase at 50° C. bytwo-fold. Similarly, the chaperone activity of αB-crystallin preventedunfolding and aggregation of citrate synthase at 45° C. Chemicalchaperones, such as glycerol, trehalose and trimethylamine-N-oxide, canalso be used for protein stabilization at a moderately high temperatureor in the presence of denaturants. The ligand binding of proteins oftenenhances stability by virtue of coupling of binding with unfoldingequilibrium. For instance, binding of biotin to streptavidin andanilinonaphthalene sulphonate derivatives to bovine serum albuminincreased T_(m) values of these proteins. The effect of calcium bindingon stability of serine protease, subtilisin S41 from the AntarcticBacillus, also has been reported.

Improvements in stability also have been accomplished by chemicalmodification and immobilization. Chemical modification andimmobilization have been used for improving protein stability byreducing conformational flexibility. For example, glycosidation ofphenylalanine dehydrogenase with cyclodextrin derivatives enhanced itsstability. Immobilization of penicillin G acylase on glyoxyl-agarosesupports via lysine-mediated coupling improved its stability. Inaddition, reduced conformational flexibility can also be achieved bycross-linking the N- and C-termini of a target protein. For instance,beta lactamase and dihydrofolate reductase with their respective N- andC-termini connected through backbone cyclization were slightly morestable than the wild-type ones.

Previous methods of stabilizing proteins do have limitations. Enhancedstabilization achieved by rational, combinatorial and data-driven designinvolves changes in residues of a target protein usually in the form ofmutations and recombinations. These changes very often compromiseintrinsic properties of proteins, such as activity and specificity. Thisalso occurs with chemical modification of proteins and theirimmobilization. Reduced conformational flexibility by modification andimmobilization usually result in the significant loss of enzymaticactivity. Recently, comprehensive directed evolution studies havedemonstrated that stability and activity are not always inverselycorrelated. For instance, directed evolution of phosphate dehydrogenaseled to identification of the variant with improved stability andactivity. However, mutations and recombinations that improve stabilitywith no compromise in activity or specificity are very rare anddifficult to predict. This limitation would be even worse forstabilization of proteins with discontinuous catalytic domains. Mutationof residues to those commonly found in naturally existing stablecounterparts improved stability of mesophilic proteins with no activityloss. However, only a small fraction of thermophilic proteins in naturehave been identified. Also, a thermophilic protein with desiredproperties (such as activity and selectivity) is not always availablefrom naturally existing ones. Employment of chaperones for stabilizationis not very practical due to their lack of specificity and requirementof a relatively large dose. Stabilization by ligand addition requirestight binding (or the presence of excess ligands), which is not alwaysavailable in normal proteins.

Therefore, it can be seen that new methods for the stabilization ofproteins can be advantageous. It also can be seen that new methods forthe stabilization of proteins that without modification of their primarysequence can be advantageous. The present invention is directed to suchnew methods and others.

BRIEF SUMMARY OF THE INVENTION

Insufficient stability of proteins is a fundamental problem thatrestricts their application in many areas. Although several strategieshave been reported to improve protein stability, an approach that worksfor a specific protein may not always work for others. The conventionalmethod for protein stabilization involves mutagenesis and thereforerisks alteration of a protein's desired properties, such as activity andspecificity. The present invention is a novel and potentially generalmethod for the stabilization of target protein domains without anymodification of their primary sequence. The method of the presentinvention employs domain insertion of a target protein into athermophilic scaffold protein. Insertion of a model target protein,exoinulinase (EI), into a loop of a thermophilic maltodextrin-bindingprotein from Pyrococcus furiosus (PfMBP) resulted in improvement ofkinetic stability of the EI domain without any compromise in itsactivity. Insertion of TEM-1 beta lactamase (BLA) at this same site inPfMBP stabilized BLA without altering its substrate specificity,suggesting that the described method can potentially be applied to awide range of proteins.

It is anticipated that the described methodology for improving proteinstability with little or no compromise in intrinsic properties will bedirectly relevant to a host of other systems, including enzymaticbiodiesel/bioethanol production, enzymatic synthesis oforganic/polymeric materials, immobilization of a protein on surfaces andemployment of a protein for therapeutic purposes.

Limited stability is a common problem associated with most proteins. Theresults of the rational, combinatorial and data-driven design of highlystable proteins have revealed the presence of different ways forstabilization. This underscores the difficulty in developing a generalstrategy of enhancing the protein stability and the importance ofindividual structural contexts in the success of conventionalstabilization methods. Enhanced stabilization achieved by theseconventional methods involves changes in side chains of target proteinresidues usually in the form of mutations and recombinations. Thesechanges very often compromise intrinsic properties of proteins. Usuallymutations and recombinations that improve stability with no compromisein activity or specificity are very rare and difficult to predict.Mutation of residues to those commonly found in naturally existingstable counterparts improved stability of mesophilic proteins with noactivity loss. However, only a small fraction of thermophilic proteinsin nature have been identified and natural thermophilic proteins withdesired activity and selectivity are not always available.

In order to improve stability of a protein with no change in side chainsof its residues, we employ a thermophilic protein as a robust scaffoldwith which a target protein domain is fused. Two possible modes ofconnection exist, “end-to-end” or “insertional” fusion. In general, theend-to-end fusion of two distinct proteins, in which the N-terminus ofone protein is connected to the C-terminus of the other, keeps thefunctions of both proteins unchanged. On the other hand, the insertionalfusion, where one protein is inserted into the middle of the other,often produces functional “cross-talking” between the proteins. Asinsertion involves more than one connection, the resultant fusionprotein is expected to form a more stable structure if an insertion siteis properly selected. For these reasons, we believe that some specificinsertion modes of a target protein into a thermophilic protein couldimprove target protein-associated (thermo)stability. The insertionalfusion can be readily achieved in both site-specific and random waysusing recombinant DNA techniques.

A thermophilic maltose binding protein from Pyrococcus furiosus (PfMBP)was chosen as a stabilizing scaffold protein. Currently available 3Dstructural information on PfMBP is also useful for the selection of aninsertion site. Insertion of the entire protein domain into anotherprotein often results in generation of a nonfunctional protein complexand structural modeling to predict successful domain insertion sites isvery challenging. Loop-forming residues 125-126 of PfMBP instead wereselected as the initial insertion site as a loop region of a givenprotein is in general tolerant to modifications, such as mutations andinsertions, among many other structural units.

Exoinulinase from Bacillus sp. Snu-7 (EI) was chosen as an initial modeltarget protein. EI is a 450-residue glycoside hydrolase catalyzingrelease of the terminal fructose from the non-reducing end of inulin. Weinserted the wild-type EI between residues 125 and 126 of PfMBP tocreate a protein complex named PfMBP-EI125 and measured its EI activityat 37° C. PfMBP-EI125 displayed nearly the same activity as thewild-type EI (the ratio of activity of the PfMBP-EI125 to the wild-typeEI=0.96±0.04). Kinetic stabilities of the wild-type EI and PfMBP-EI125were evaluated by measuring their respective activities over the timeduring incubation at 37° C. Interestingly, the kinetic stability at 37°C. of PfMBP-EI125 was much higher than that of the wild-type EI. Thetime-course activities of the wild-type EI followed a second-orderinactivation (R2>0.94). Consistent with the second-order inactivationkinetics, the formation of EI precipitate was observed after 20 dayincubation of the wild-type EI. As a result, the concentration of EI inthe solution was significantly reduced after a 20 day incubation. Noprecipitation was however observed in PfMBP-EI125 and its concentrationin the solution remained the same after the 20 day incubation. The orderof inactivation kinetics of PfMBP-EI125 could not be determined becauseno sufficient activity loss during the given incubation at 37° C. wasobserved. A similar decay was observed in circular dichorism andintrinsic tryptophan fluorescence of the wild-type EI and PfMBP-EI125,indicating that their secondary and tertiary structures changed at thesame rate.

The connection between PfMBP and EI created by domain insertion shouldcause proximity of these two proteins, which may allow stronginteractions between those. To test whether the proximity of proteindomains created by domain insertion is required for stabilization,kinetic stability of the wild-type EI mixed with the purified PfMBP wasevaluated. Coincubation with PfMBP yielded no significant improvement inkinetic stability of EI. The EI precipitate was also formed from thesample containing EI coincubated with PfMBP after 20 days at 37° C.These indicate that stabilization was achieved by the specific linkagebetween EI and PfMBP, not by non-specific effects caused by the presenceof PfMBP. Whether the observed stabilization effect of domain insertioncan be achieved by end-to-end connection, employed for construction ofPfMBP-EI381, was examined. The end-to-end connection between PfMBP andEI within PfMBP-EI381 yielded a low initial activity (˜35% of thewild-type EI) and no improvement of kinetic stability. The formation ofEI precipitate was also observed after 20 day incubation of PfMBP-EI381at 37° C. These data suggest that only insertional, not end-to-end,fusion improved kinetic stability of EI.

To examine whether insertion into PfMBP would also be effective at thestabilization of other proteins, a 268-residue TEM 1 beta-lactamase(BLA) was inserted into PfMBP at the site between residues 125 and 126(the resultant protein complex named PfMBP-BLA125). PfMBP-BLA125 showed˜42% of the wild-type BLA activity at 25° C. The time-course activitiesof these proteins during incubation at 25° C. followed a first-orderinactivation. PfMBP-BLA125 showed a lower value of the observedfirst-order inactivation constant, kobs1, than the wild-type BLA,indicating that insertion of the wild-type BLA into PfMBP slowed theirreversible loss of BLA activity at 25° C. This result is in contrastwith the consequence of domain insertion into a mesophilic maltosebinding protein from Escherichia coli (EcMBP) at the site correspondingto residues 125 and 126 of PfMBP. Structures of PfMBP and EcMBP wereclosely superimposed. Insertion of BLA and a 13 aa peptide sequence intoEcMBP at the site between residues 120 and 121, identified throughstructural superposition to be corresponding to residues 125 and 126 ofPfMBP8, resulted in formation of inclusion bodies in previous studies.These data suggest that nature of a scaffold protein may determinefolding and intracellular stability of a protein insertion complex. Thesubstrate specificity of the wild-type BLA and PfMBP-BLA125 was measuredby evaluating an apparent second-order rate constant, kcat/Km, at 25° C.for different substrates, nitrocefin and cefotaxime. The ratio ofkcat/Km values for nitrocefin to cefotaxime were similar, suggestingthat the substrate specificity of the BLA domain can be largelymaintained after insertion into PfMBP.

Herein, we show that domain insertion into PfMBP can significantlyimprove kinetic stability of EI and BLA. The same insertion site waseffective at enhancing stability of both EI and BLA, suggesting thepotential generality of the described method. Unlike conventionalstabilization methods, the approach described herein does not requireany change on a target protein except for its connection to the scaffoldprotein. As a result, intrinsic properties of a target protein, such asactivity and specificity, can be largely maintained.

These features, and other features and advantages of the presentinvention will become more apparent to those of ordinary skill in theart when the following detailed description of the preferred embodimentsis read in conjunction with the appended figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Amino acid sequences of PfMBP-EI125, PfMBP-EI381 andPfMBP-BLA125. The numbers indicate the amino acid numbers of givenproteins. The signal sequence of EcMBP was included for all proteincomplexes to enable export to the periplasm. Residues 1-23 of BLA(corresponding to its signal sequence) were not included in the fusionsas this sequence is not present in the mature BLA.

FIG. 2: Time-course changes in (a) exoinulinase activity, (b)ellipticity at 220 nm and (c) tryptophan fluorescence of the wild-typeEI (□), PfMBP-EI125 (Δ), PfMBP-EI381 (⋄), and an equimolar mixture ofPfMBP+the wild-type EI (∘) during incubation at 37° C. The proteinconcentration of the wild-type EI, PfMBP, PfMBP-EI125 and PfMBP-EI381 inthe assay buffer was 1 μM. (a) Activity was measured using 500 μM ofinulin. Activity of each protein sample at time zero was set to 100%.Activities of the wild-type EI, PfMBP-EI125 and PfMBP+the wild-type EIwere all similar at the beginning of incubation. The errors representone standard deviation (n≧3). (b) and (c), Ellipticity and tryptophanfluorescence at time zero was set to 100% for each protein sample.Excitation wavelength was 280 nm and emission was monitored at 337 nm.

FIG. 3: The amount of soluble proteins during incubation at 37° C.Protein samples were centrifuged after 0 and 20-day incubation at 37°C., and the supernatants were loaded in a SDS-PAGE gel. All samplescontained 1 μM of proteins. M: molecular weight marker. 1: PfMBP-EI125at the beginning of incubation. 2: PfMBP-EI125 after 20-day incubation.3: the wild-type EI at the beginning of incubation. 4: the wild-type EIafter 20-day incubation. *: PfMBP-EI125. **: the wild-type EI.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Limited stability is a common problem associated with many proteins. Theresults of rational, combinatorial and data-driven design of highlystable proteins have revealed different paths for stabilization. Thisunderscores the difficulty in developing a general strategy forenhancing protein stability and the importance of individual structuralcontexts in the success of conventional stabilization methods. Enhancedstabilization achieved by these conventional methods involves changes inside chains of target protein residues usually in the form of pointmutations. These changes very often compromise a protein's intrinsicproperties. Rational and directed evolution approaches can sometimesresult in identification of protein variants with improved stabilitywithout compromised activity. However, mutations that improve stabilitywith no compromise in activity or specificity are in general very rareand difficult to predict, especially for a large protein with multiplediscontinuous catalytic domains. Furthermore, such mutation-basedmethods must be optimized for every specific target, as there are nogeneral rules for protein stabilization. Mutation of residues to thosecommonly found in naturally existing thermostable counterparts canimprove stability of mesophilic proteins without activity loss. However,only a small fraction of thermophilic proteins in nature have beenidentified and natural thermophilic proteins with desired activity andselectivity are not always available.

In order to improve the stability of a protein without changing itsprimary sequence, a thermophilic protein is employed as a robustscaffold to which a target protein domain is fused. Two possible modesof connection exist, “end-to-end” and “insertional” fusion. Inend-to-end fusion, the N-terminus of one protein is connected to theC-terminus of the other. Unlike end-to-end fusion, insertional fusion,in which one protein is inserted into the middle of the other, oftenproduces functional “cross-talk” between the proteins. As insertioninvolves more than one connection, the resultant fusion protein has thepotential to form a more stable structure if an insertion site isproperly selected. For these reasons, it is speculated that somespecific insertion modes of a target protein into a thermophilic proteincould improve target protein-associated stability. Insertional fusioncan be readily achieved by site-specific and random methodologies usingrecombinant DNA techniques. The close proximity of the N- and C-terminiof an inserted protein seems to increase the chance of successfulinsertion. Nearly 50% of single-domain proteins have their N- andC-termini proximal, indicating the potential application of the presentmethod to a wide range of proteins. The incorporation of appropriatelinkers between the protein domains can assist in achieving functionalinsertion and help accommodate insertion of proteins whose two terminiare more distal.

A maltodextrin-binding protein from the hyper-thermophile Pyrococcusfuriosus (PfMBP) is a 43 kDa periplasmic protein and highly stableagainst heat and chemical denaturation. For instance, PfMBP displayslittle loss in a secondary structure at 85° C. or in 6M guanidinehydrochloride whereas the mesophilic maltodextrin-binding protein fromEscherichia coli (EcMBP) unfolds at 65° C. or in 1M guanidinehydrochloride. PfMBP was chosen as the stabilizing scaffold protein forseveral reasons. First, end-to-end fusion to maltodextrin-bindingproteins from bacteria and archaea often stabilizes the fusion partnerby increasing its solubility and preventing the formation of inclusionbodies. The thermophilic nature of PfMBP might allow exploring theinsertion sites, which would be unavailable in a mesophilic protein dueto limited stability. Second, 3D structural information on PfMBP isavailable. The loop-forming residues 125 and 126 of PfMBP were selectedas the initial insertion site (FIG. 1), as surface loops are in generalmore tolerant to mutations and insertions than other structural units.

Exoinulinase from Bacillus sp. Snu-7 (EI) was chosen as an initial modeltarget protein as it has limited stability at 37° C. EI is a 495-residueglycoside hydrolase catalyzing release of the terminal fructose from thenon-reducing end of inulin. Wild-type EI lost activity irreversiblyduring incubation at 37° C. (FIG. 2 a). The time-course activities ofthe wild-type EI followed a second-order inactivation (R²>0.94).Consistent with the second-order inactivation kinetics, the formation ofprecipitate was observed after a 20-day incubation of the wild-type EI(data not shown). As a result, the concentration of soluble EI wassignificantly reduced after the 20-day incubation (FIG. 3).

The wild-type EI was inserted between residues 125 and 126 of PfMBP tocreate a protein complex named PfMBP-EI125 (FIG. 1—top) and measured itsexoinulinase activity at 37° C. PfMBP-EI125 displayed nearly the sameactivity as the wild-type EI. The ratio of activity of PfMBP-EI125 towild-type EI was 0.96±0.04. Kinetic stability of PfMBP-EI125 wasevaluated by measuring their respective activities as a function of timeduring incubation at 37° C. As desired, the kinetic stability ofPfMBP-EI125 was much higher than that of the wild-type EI (FIG. 2 a). Noprecipitation was observed in solutions of PfMBP-EI125 (data not shown)and its concentration in solution remained unchanged after a 20-dayincubation at 37° C. (FIG. 3). The order of inactivation kinetics ofPfMBP-EI125 could not be determined due to minimal activity loss duringincubation. The decay in both proteins' circular dichroism and intrinsictryptophan fluorescence spectra mirrored the loss in activity (FIGS. 2 band c), suggesting that changes in secondary and tertiary structureswere responsible for the loss in activity.

To test whether fusion of the protein domains was required forstabilization, the kinetic stability of an equimolar mixture ofwild-type EI and PfMBP was evaluated. Coincubation with PfMBP at anequimolar ratio yielded no significant improvement in the kineticstability of wild-type EI (FIG. 2) and did not prevent the precipitationobserved after 20 days (data not shown). This indicates thatstabilization was achieved by the linkage between EI and PfMBP, not bynon-specific effects caused by the presence of PfMBP. Whether theobserved stabilization effect of domain insertion could be achieved byend-to-end fusion was next examined. The end-to-end fusion between PfMBPand EI domains within PfMBP-EI381 (FIG. 1—middle) resulted in reducedinitial activity (˜35% of the wild-type EI) and no improvement ofkinetic stability (FIG. 2). The formation of precipitate was alsoobserved after a 20-day incubation of PfMBP-EI381 at 37° C. (data notshown). Thus, simple end-to-end fusion was not sufficient to improve thekinetic stability of EI, implying there is something special about theinsertional fusion. This may include specific relative orientations ofEI and PfMBP domains in the insertional fusion that favor inter-domaininteractions stabilizing the inserted protein, and/or the presence oftwo “tethers” between the domains in the insertional fusion. The resultalso suggests that the improved stability of the EI domain achieved byinsertional fusion was not merely due to enhancement of solubility uponfusion to PfMBP, which has been known to prevent inclusion bodyformation of end-to-end fusion partners.

To examine whether insertion into PfMBP would be effective for thestabilization of other proteins, the 263-residue TEM 1 beta-lactamase(BLA) was inserted into PfMBP at the same site that EI was inserted inPfMPB-EI125 (the resultant fusion protein named PfMBP-BLA125, FIG.1—bottom). PfMBP-BLA125 showed ˜42% of the wild-type beta-lactamaseactivity at 25° C. (Table 1). The time-course activities of thewild-type BLA and PfMBP-BLA125 during incubation at 25° C. followed afirst-order inactivation (R²>0.94). PfMBP-BLA125 showed a 35% lowerobserved first-order inactivation constant, k_(obs1), than the wild-typeBLA (Table 1), indicating that insertion of the wild-type BLA into PfMBPslowed the irreversible loss of beta-lactamase activity at 25° C. Takentogether with the EI result, this suggests that insertional fusion toPfMBP could improve stability of proteins following various inactivationmechanisms, for example, first and second-order irreversibledenaturation. The substrate specificity of the wild-type BLA andPfMBP-BLA125 was measured by evaluating an apparent second-order rateconstant, k_(cat)/K_(m), at 25° C. for the substrates, nitrocefin (NCF)and cefotaxime (CFTX). The ratio of k_(cat)/K_(m) values for NCF to CFTXwere similar for both proteins (Table 1), suggesting that the substratespecificity of the BLA domain can be largely maintained after insertioninto PfMBP.

A comparison of results of the present method with that of previousinsertion studies into EcMBP suggests that a thermophilic scaffolddomain is required for the inserted domain to acquire improvedstability. The structures of PfMBP and EcMBP closely superimpose andresidues 120 and 121 of EcMBP are structurally aligned with residues 125and 126 of PfMBP. Insertion of the wild-type BLA into EcMBP betweenresidues 120 and 121 resulted in the formation of inclusion bodies inprevious studies. The formation of inclusion bodies was also observed ininsertion of a short 13-aa peptide sequence into EcMBP at the same site.This suggests that inclusion body formation could primarily be due toincomplete folding of EcMBP, and the high stability displayed by PfMBPmight allow for insertion into the sequence space, which is notavailable in the moderately stable EcMBP. Overall, the nature of ascaffold protein may determine folding and intracellular stability of aprotein insertion complex.

TABLE 1 Kinetic and thermodynamic parameters of the wild-type BLA,PfMBP-BLA125 and PfMBP-BLA125 DKS at 25° C. The wild-type PfMBP- PfMBP-BLA^(a) BLA125^(a) BLA125DKS^(a) Relative activity at 100 ± 12  42 ± 3 61 ± 3  t = 0^(b) k_(obs1) ^(c,d) [h⁻¹] 0.31 ± 0.02 0.20 ± 0.01 0.19 ±0.01 (k_(cat)/K_(m))_(NCF) ^(e) 10 ± 2  5 ± 1 6 ± 2 (s⁻¹ μM⁻¹)(k_(cat)/K_(m))_(CFTX) 2.5 ± 0.2 1.0 ± 0.1 1.5 ± 0.2 (s⁻¹ mM⁻¹)(k_(cat)/K_(m))_(NCF)/ 4,000 ± 1,000 5,000 ± 1,500 4,000 ± 1,500(k_(cat)/K_(m))_(CFTX) ^(e) ^(a)Samples contained 5 nM of proteins^(b)Beta-lactamase activity of the wild-type BLA was set to 100%.Activity was measured using 100 μM of nitrocefin. ^(c)k_(obs1) wasevaluated by fitting reaction velocities of nitrocefin hydrolysismeasured at several time points during incubation at 25° C. tofirst-order irreversible inactivation kinetics. ^(d)The error representsa 95% confidence level evaluated from linear regression using Polymath.^(e)The error was evaluated by the propagation of error methods. Othererrors represent one standard deviation (n ≧ 3)

In the present invention, it has been found that domain insertion of EIand BLA into PfMBP can significantly improve their kinetic stability.The same insertion site was effective at enhancing stability of both EIand BLA, suggesting the potential generality of the described method.Unlike conventional stabilization methods, the approach described heredoes not require any change on a target protein except for itsconnection to the scaffold protein. As a result, the intrinsicproperties of a target protein, such as activity and specificity, can belargely maintained.

Incorporation of inter-domain linkers. It is reasoned that the correctgeometric arrangements of PfMBP and BLA and conformational flexibilityin the connection between these two domains could restore thecompromised activity found in PfMBP-BLA125 by reducing mutualinterference between folding of each domain. To test this, peptidyllinkers, DKS, were introduced between BLA and PfMBP domains withinPfMBP-BLA125. Incorporation of linkers in PfMBP-BLA125DKS improvedinitial BLA activity while maintaining kinetic stability compared toPfMBP-BLA125 (Table 1). The ratio of apparent second-order rate constantvalues, k_(cat)/K_(m), for different substrates, nitrocefin andcefotaxime, of the wild-type BLA and PfMBP-BLA125DKS were similar,suggesting that the substrate specificity of the BLA domain can belargely maintained after insertion into PfMBP and incorporation oflinkers (Table 1).

Materials and Methods Reagents

Oligonucleotides were purchased from Operon Biotechnologies Inc.(Huntsville, Ala., USA). High fidelity platinum pfx DNA polymerase andElectromax DH5α-E cells were purchased from Invitrogen (Carlsbad,Calif., USA). All DNA purification kits were purchased from Qiagen(Valencia, Calif., USA). His-tag protein purification kits and columnswere purchased from Novagen (Madison, Wis., USA) and GE healthcare(Buckinghamshire, England, UK), respectively. Restriction enzymes and T4DNA ligase were purchased from New England Biolabs, Inc. (Ipswich,Mass., USA). Nitrocefin (NCF) was purchased from Remel (Lenexa, Kans.,USA). Cefotaxime (CFTX) was purchased from Sigma (St. Louis, Mo., USA).Inulin, all other antibiotics and biological reagents were purchasedfrom Thermo Fisher Scientific (Waltham, Mass., USA).

DNA Construction

The plasmid, pREX12, for expression of the wild-type exoinulinase (EI)was kindly provided by Dr. S. I. Kim (Seoul National University, Seoul,Korea). PCR was used for replicating DNA sequences coding for the entiremaltodextrin-binding protein from Pyrococcus furiosus (PfMBP) fromplasmid FLIPmal_Pf generously provided by Dr. W. B. Frommer (CarnegieInstitute of Plant Biology, Stanford, Calif., USA) and the entire TEM-1beta lactamase (BLA) from plasmid pBR322 (Fermentas, Glen Burnie, Md.,USA). A six histidine tag was genetically attached to the C-termini ofPfMBP and BLA, for protein purification. The signal sequence ofmaltodextrin-binding protein from Escherichia coli (EcMBP) (residue1-30) was added to PfMBP for export of the protein to the periplasm ofE. coli. Sequences of PfMBP and EcMBP were aligned beginning with thesixth residue of PfMBP (numbered according to Evdokimov et al.). Thedesired PCR products were purified by QIAquick PCR purification andQIAquick gel extraction kits.

For construction of PfMBP-EI125, PfMBP-EI381 and PfMBP-BLA125, DNAsequences coding for the wild-type EI and BLA, and parts of PfMBP wereamplified by PCR from pREX12, pBR322 and FLIPmal_pf, respectively. Thepurified DNA fragments were assembled into a full gene by overlapextension PCR. A six histidine tag was genetically added to theC-terminus of each fusion complex. The signal sequence of EcMBP wasincluded in PfMBP-EI125, PfMBP-EI381 and PfMBP-BLA125. No additionallinker was added between protein domains.

The DNA sequences coding for PfMBP, the wild-type BLA, PfMBP-EI125,PfMBP-E1381 and PfMBP-BLA125 were digested by BamHI and SpeI restrictionenzymes to create sticky ends needed for ligation. Plasmid pDIM-C8-MalEwas digested with BamHI and SpeI restriction enzymes, and purified byQIAquick gel extraction kit. The digested inserts and plasmids were thenligated using T4 ligase and supplied buffer. Ligation products were thenelectroporated into 40 μl Electromax DH5α-E using a Bio-Rad Gene Pulser(Hercules, Calif., USA). Electroporated cells were subsequentlyincubated for 1 hour at 250 rpm and 37° C. in a New Brunswick ScientificInnova TM4230 incubator (Edison, N.J., USA). Electroporated cells werethen plated on LB agar plate supplemented with 50 μg/ml chloramphenicoland incubated for 16-24 hours at 37° C. in the incubator. The coloniesgrowing on LB agar plate supplemented with 50 μg/mlchloramphenicol werepicked and recultured in test tubes containing 10 ml LB media and 50μg/ml chloramphenicol. Plasmid DNA was extracted from reculturedcolonies using QIAprep spin miniprep kit according to the manufacturer'sprotocol. The extracted DNA was then sequenced at Genewiz, Inc. (SouthPlainfield, N.J., USA).

Protein Expression and Purification

One liter of LB media containing 50 μg/ml chloramphenicol was inoculatedwith 2% overnight culture and shaken at 250 rpm at 37° C. Cellsexpressing the wild-type EI, PfMBP, PfMBP-EI125 and PfMBP-EI381 weregrown at 37° C. until the optical density at 600 nm was 0.6. Expressionof the wild-type EI, PfMBP, PfMBP-EI125 and PfMBP-EI381 was then inducedby adding 1 mM isopropyl-beta-D-1-galactopyranoside (IPTG). Afterinduction, the cell culture was shaken at 250 rpm for another 16-24hours at 23 or 30° C. Cells were pelleted by centrifuging at 5000 rpm at4° C. for 20 minutes using a Beckman Coulter Avanti JE centrifuge(Fullerton, Calif., USA). The pelleted cells were then stored at −75° C.until ready for use. For protein purification, the pelleted cells wereresuspended in 0.05 M Tris-HCl buffer containing 0.5 M NaCl, pH 7.5 witha dilution ratio of approximately 10 ml per gram of cells. The cellswere then lysed by French Press purchased from Thermo Fisher Scientificand the cell lysates were centrifuged at 20,000 rpm at 4° C.Supernatants containing the soluble proteins were then recovered andpassed over the Ni²⁺ column. Bound proteins were eluted with imidazolesolution and dialyzed at 4° C. against at least fifteen liter of 0.05 MTris-HCl buffer, pH 7.5. Purified protein samples were stored at 4° C.

The wild-type BLA and PfMBP-BLA125 were expressed and purified in thesame way as described above except for the use of 0.05 M Na₂HPO₄/NaH₂PO₄containing 0.1 M NaCl, pH 7.2 as a resuspension buffer. The proteineluted with imidazole solution was dialyzed at 4° C. against the samebuffer. Solution containing the wild-type BLA and PfMBP-BLA125 weresubject to the second dialysis against the same buffer containing 20%glycerol, then aliquoted and stored at −20° C.

The purities of the proteins were estimated by Coomassie Blue stainingof sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)and were greater than 95%. Protein concentrations were determined usingextinction coefficients at 280 nm as calculated according to Gill andvon Hippel or the Bradford assay.

Enzyme Assay

Hydrolysis of inulin by the wild-type EI, an equimolar mixture ofPfMBP+the wild-type EI, PfMBP-EI125 and PfMBP-EI381 was measured in 0.05M Tris-HCl buffer, pH 7.5 at 37° C., as described previously. Theprotein concentration of the wild-type EI, PfMBP, PfMBP-EI125 andPfMBP-EI381 in the assay buffer was 1 μM. Protein samples were incubatedat 37° C. prior to addition of inulin. The final concentration of inulinwas 500 μM in all assays. For the measurement of inulin hydrolysis, areaction mixture containing a protein and inulin was incubated at 37° C.for additional one hour followed by addition of 3,5-dinitosalicylicacid. The reaction mixture was then boiled for 10 min and an amount ofliberated reducing sugar was determined by absorbance at 550 nm aspreviously described.

Enzymatic hydrolysis of nitrocefin (NCF) was measured with the wild-typeBLA and PfMBP-BLA125 at 25° C. in 0.1 M Na₂HPO₄/NaH₂PO₄ buffercontaining 0.1 M NaCl, pH 7.2, as described previously. Proteinconcentrations of the wild-type BLA and PfMBP-BLA125 were 5 nM in theassay buffer. NCF was added to protein samples incubated at 25° C. Thefinal concentration of NCF was 100 μM in all assays. The initial rate ofNCF hydrolysis was measured by monitoring changes in absorbance at 486nm over time using a Varian Cary 50 UV-VIS spectrophotometer (Palo Alto,Calif., USA) fitted with a Quantum Northwest Peltier temperature controlunit (Shoreline, Wash., USA).

In order to monitor irreversible inactivation of proteins over time,protein samples were withdrawn at different time points of incubationand their enzymatic hydrolyses were measured at the incubationtemperature.

Evaluation of Michaelis-Menten Kinetic Parameters

Enzymatic hydrolysis of NCF was measured as described above. TheMichaelis-Menten parameters, k_(cat) and K_(m), for NCF hydrolysis werethen determined from the initial rate of enzymatic hydrolysis usingEadie-Hofstee plots. Molecular extinction coefficient change of NCF uponits hydrolysis (Δε_(NCF)) was reported to be 17420 M⁻¹cm⁻¹. Thewild-type BLA that was prepared showed Michaelis-Menten kineticparameters for NCF hydrolysis comparable to those previously reported(k_(cat)=800±40 s⁻¹ and K_(m)=80±8 μM at 25° C. in the current study vsk_(cat)=900 s⁻¹ and K_(m)=110 μM at 24° C. from Sigal et al.). Forcefotaxime (CFTX) hydrolysis, absorbance at 260 nm was monitored as afunction of time. The value of k_(cat)/K_(m) was determined fromsteady-state rates with 80 μM of CFTX, which is quite low compared toK_(m) for cefotaxime. K_(m) for cefotaxime hydrolysis by the wild-typeBLA and PfMBP-BLA125 was determined to be >1 mM using a competitiveassay with 100 μM NCF as described previously. Molecular extinctioncoefficient change of CFTX upon its hydrolysis (Δε_(CFTX)) was reportedto be 6510 M⁻¹cm⁻¹. The wild-type BLA we prepared showed the similark_(cat)/K_(m) value for CFTX hydrolysis to that previously reported(k_(cat)/K_(m) at 25° C.=2.5±0.2 s⁻¹ mM⁻¹ in the current study vsk_(cat)/K_(m) at 30° C.=2.2 s⁻¹ mM⁻¹ from Sowek et al.).

Evaluation of the First-Order Irreversible Inactivation Constant

The fitting of the time-course activity data to the irreversibleinactivation kinetics of various orders was attempted. The inactivationkinetics of the wild-type BLA and PfMBP-BLA125 were determined to be thefirst-order based on R² values of fitting (R²>0.94). The first-orderirreversible inactivation constants (k_(obs1)) were then determined byfitting the data to the equation, d[E]/dt=−k_(obs1)[E] where [E]=theinitial rate of enzymatic NCF hydrolysis after incubation of a proteinat 25° C. for time t). The 95% confidence level of k_(obs1) wasevaluated using Polymath.

The second-order irreversible inactivation kinetics was able to capturethe activity loss over time of the wild-type EI, PfMBP-EI381 and anequimolar mixture of PfMBP+the wild-type EI (R²>0.94), but notPfMBP-EI125. This is because PfMBP-EI125 did not display sufficientactivity loss during the given incubation time period at 37° C.Therefore, no further attempt to determine the irreversible inactivationconstants of exoinulinase activity was made.

Circular Dichroism Spectroscopy

Secondary structures of proteins were determined using circulardichroism (CD), collected using a Jasco J-815 circular spectrometer(Easton, Md., USA) in the far-UV range with a 0.1 cm pathlength ofcuvette. The protein samples were withdrawn at several time pointsduring incubation at 37° C. Ellipticity of samples containing 1 μM ofthe wild-type EI in the presence and absence of equimolar PfMBP at eachwavelength was measured without dilution at 37° C. Ellipticity ofsamples containing 1 μM of PfMBP-EI125 and PfMBP-EI381 was measured inthe same way. The spectrum of the background (buffer only) was measuredand then subtracted from the sample spectrum.

Intrinsic Tryptophan Fluorescence

Intrinsic tryptophan fluorescence of protein samples was measured usinga Photon Technology QuantaMaster QM-4 spectrofluorometer (Birmingham,N.J., USA). Excitation wavelength was 280 nm and emission was monitoredat 337 nm. The protein samples were withdrawn at several time pointsduring incubation at 37° C. Intrinsic tryptophan fluorescence of samplescontaining 1 μM of the wild-type EI with and without addition ofequimolar PfMBP was measured without dilution. Intrinsic tryptophanfluorescence of samples containing 1 μM of PfMBP-EI125 and PfMBP-EI381was measured in the same way. The spectrum of the background (bufferonly) was measured and then subtracted from the sample spectrum.

SDS-PAGE for Determination of the Amount of Soluble Proteins

Protein samples were incubated for the designated time period and thencentrifuged. The supernatant of each sample was loaded in the SDS-PAGEgel. The gel was then stained with Coomassie Blue.

Thus, using the above disclosed illustrative materials and methods, ithas been found that domain insertion of EI and BLA into PfMBP cansignificantly improve their kinetic stability. The same insertion sitewas effective at enhancing stability of both EI and BLA, suggesting thepotential generality of the described method for use in other proteins.Unlike conventional stabilization methods, the approach described hereindoes not require any change on a target protein except for itsconnection to the scaffold protein. As a result, the intrinsicproperties of a target protein, such as activity and specificity, can belargely maintained.

It is to be understood that the present invention is by no means limitedto the particular constructions and method steps herein disclosed orshown in the drawings, but also comprises any modifications orequivalents within the scope of the claims known in the art. It will beappreciated by those skilled in the art that the devices and methodsherein disclosed will find utility with respect to enzymatic reactionsemployed in the biochemical industry and with therapeutic proteins.

REFERENCES

-   A1. Eijsink, V. G., Bjork, A., Gaseidnes, S., Sirevag, R., Synstad,    B., van den Burg, B. & Vriend, G. (2004) Rational engineering of    enzyme stability. J. Biotechnol. 113, 105-20.-   A2. Eijsink, V. G., Gaseidnes, S., Borchert, T. V. & van den    Burg, B. (2005) Directed evolution of enzyme stability. Biomol. Eng.    22, 21-30.-   A3. Bommarius, A. S., Broering, J. M., Chaparro-Riggers, J. F. &    Polizzi, K. M. (2006) High-throughput screening for enhanced protein    stability. Curr. Opin. Biotechnol. 17, 606-610.-   A4. Korkegian, A., Black, M. E., Baker, D. & Stoddard, B. L. (2005)    Computational thermostabilization of an enzyme. Science 308,    857-860.-   A5. Johannes, T. W., Woodyer, R. D. & Zhao, H. M. (2005) Directed    evolution of a thermostable phosphite dehydrogenase for NAD(P)H    regeneration. Appl. Environ. Microbiol. 71, 5728-5734.-   A6. Giver, L., Gershenson, A., Freskgard, P. O. &    Arnold F. H. (1998) Directed evolution of a thermostable esterase    Proc. Natl. Acad. Sci. USA. 95, 12809-12813.-   A7. Lehmann, M. & Wyss, M. (2001) Engineering proteins for    thermostability: the use of sequence alignments versus rational    design and directed evolution. Curr. Opin. Biotechnol. 12, 371-375.-   A8. Doi, N. & Yanagawa, H. (1999) Insertional gene fusion    technology. FEBS Lett. 457, 1-4.-   A9. Ostermeier, M. (2005) Engineering allosteric protein switches by    domain insertion. Protein Eng. Des. Sel. 18, 359-364.-   A10. Krishna, M. M. & Englander, S. W. (2005) The N-terminal to    C-terminal motif in protein folding and function. Proc. Natl. Acad.    Sci. USA. 102, 1053-1058.-   A11. Evdokimov, A. G., Anderson, D. E., Routzahn, K. M. &    Waugh, D. S. (2001) Structural basis for oligosaccharide recognition    by Pyrococcus furiosus maltodextrin-binding protein. J. Mol. Biol.    305, 891-904.-   A12. Gardner, K. H., Zhang, X., Gehring, K. & Kay, L. (1998)    Solution NMR studies of a 42-kDa Escherichia coli maltose binding    protein/β-cyclodextrin complex: chemical shift assignments and    analysis. J. Am. Chem. Soc. 120, 11738-11748.-   A13. Fox, J. D., Routzahn, K. M., Bucher, M. H. &    Waugh, D. S. (2003) Maltodextrin-binding proteins from diverse    bacteria and archaea are potent solubility enhancers. FEBS Lett.    537, 53-57.-   A14. Bloom, J. D., Labthavikul, S. T., Otey, C. R. &    Arnold, F. H. (2006) Protein stability promotes evolvability. Proc.    Natl. Acad. Sci. USA. 103, 5869-5874.-   A15. Puziss, J. W., Harvey, R. J. & Bassford, P. J. Jr. (1992)    Alterations in the hydrophilic segment of the maltose-binding    protein (MBP) signal peptide that affect either export or    translation of MBP. J. Bacteriol. 174, 6488-6497.-   A16. Kim, K.-Y., Koo, B.-S., Jo, D. & Kim. S.-I. (2004) Cloning,    expression, and purification of exoinulinase from Bacillus sp.    Snu7. J. Microbiol. Biotechnol. 14, 344-349.-   A17. Betton, J. M., Jacob, J. P., Hofnung, M. &    Broome-Smith, J. K. (1997) Creating a bifunctional protein by    insertion of beta-lactamase into the maltodextrin-binding protein.    Nat. Biotechnol. 15, 1276-1279.-   A18. Martineau, P., Leclerc, C. & Hofnung, M. (1996) Modulating the    immunological properties of a linear B-cell epitope by insertion    into permissive sites of the MalE protein. Mol. Immunol. 33,    1345-1358.-   B1. Kim, K.-Y., Koo, B.-S., Jo, D. & Kim. S.-I. (2004) Cloning,    expression, and purification of exoinulinase from Bacillus sp.    Snu7. J. Microbiol. Biotechnol. 14, 344-349.-   B2. Puziss, J. W., Harvey, R. J. & Bassford, P. J. Jr. (1992)    Alterations in the hydrophilic segment of the maltose-binding    protein (MBP) signal peptide that affect either export or    translation of MBP. J. Bacteriol. 174, 6488-6497.-   B3. Evdokimov, A. G., Anderson, D. E., Routzahn, K. M. &    Waugh, D. S. (2001) Structural basis for oligosaccharide recognition    by Pyrococcus furiosus maltodextrin-binding protein. J. Mol. Biol.    305, 891-904.-   B4. Kim, J. R. & Ostermeier, M. (2006) Modulation of effector    affinity by hinge region mutations also modulates switching activity    in an engineered allosteric TEM1 beta-lactamase switch. Arch.    Biochem. Biophys. 446, 44-51.-   B5. Gill, S. C. & von Hippel, P. H. (1989) Calculation of protein    extinction coefficients from amino acid sequence data. Anal.    Biochem. 182, 319-326.-   B6. Bradford, M. M. (1976) A Rapid and Sensitive Method for the    Quantitation of Microgram Quantities of Protein Utilizing the    Principle of Protein-Dye Binding. Anal. Biochem. 72, 248-254.-   B7. Melius, P. (1971) Isolation of yeast invertase by sephadex gel    chromatography. A biochemistry laboratory experiment. J. Chem. Edu.    48, 765-766.-   B8. McManus-Munoz, S. & Crowder, M. W. (1999) Kinetic mechanism of    metallo-beta-lactamase L1 from Stenotrophomonas maltophilia.    Biochemistry 38, 1547-1553.-   B9. Sigal, I. S., DeGrado, W. F., Thomas, B. J. & Petteway, S. R.,    Jr. (1984) Purification and properties of thiol beta-lactamase. A    mutant of pBR322 beta-lactamase in which the active site serine has    been replaced with cysteine. J. Biol. Chem. 259, 5327-5332.-   B10. Kumar, S., Adediran, S. A., Nukaga, M. & Pratt R F. (2004)    Kinetics of turnover of cefotaxime by the Enterobacter cloacae P99    and GCl beta-lactamases: two free enzyme forms of the P99    beta-lactamase detected by a combination of pre- and post-steady    state kinetics. Biochemistry 43, 2664-2072.-   B11. Sowek, J. A., Singer, S. B., Ohringer, S., Malley, M. F.,    Dougherty, T. J., Gougoutas, J. Z. & Bush, K. (1991) Substitution of    lysine at position 104 or 240 of TEM-1pTZ18R beta lactamase enhances    the effect of serine-164 substitution on hydrolysis or affinity for    cephalosporins and the monobactam aztreonam. Biochemistry 30,    3179-3188.-   C1. Gübitz, G. M. and Paulo, A. G. (2003) New substrates for    reliable enzymes: enzymatic modification of polymers. Curr. Opin.    Biotechnol. 14, 577-582.-   C2. Jaeger, K. E. and Eggert, T. (2002) Lipases for Biotechnology.    Curr. Opin. Biotechnol. 13, 390-397.-   C3. Kobayashi, S. and Uyama, H. (2002) In vitro polyester synthesis    via enzymatic polymerization. Curr. Org. Chem. 6, 209-222.-   C4. Daniel, R. M., Cowan, D. A., Curran, M. and Morgan, H. W. (1982)    A correlation between protein thermostability and susceptibility to    proteolysis. Biochem. J. 207, 641-644.-   C5. Owusu, R. K. and Cowan, D. A. (1989) A correlation between    microbial protein thermostability and resistance to denaturation in    aqueous-organic solvent two-phase systems. Enz. Microb. Technol. 11,    568-574.-   C6. Cowan, D. A. (1997) Thermophilic proteins: stability and    function in aqueous and organic solvents. Comp. Biochem. Physiol. A    Physiol. 118, 429-438.-   C7. Eijsink, V. G., Bjork, A., Gaseidnes, S., Sirevag, R., Synstad,    B., van den Burg, B. and Vriend, G. (2004) Rational engineering of    enzyme stability. J. Biotechnol. 113, 105-20.-   C8. Van den Burg, B., Vriend, G., Veltman, O. R., Venema, G. and    Eijsink, V. G. (1998) Engineering an enzyme to resist boiling. Proc.    Natl. Acad. Sci. USA. 95, 2056-2060.-   C9. Hasegawa, J., Shimahara, H., Mizutani, M., Uchiyama, S., Arai,    H., Ishii, M., Kobayashi, Y., Ferguson, S. J., Sambongi, Y. and    Igarashi, Y. (1999) Stabilization of Pseudomonas aeruginosa    cytochrome c551 by systematic amino acid substitutions based on the    structure of thermophilic Hydrogenobacter thermophilus cytochrome    c552. J. Biol. Chem. 274, 37533-37537.-   C10. Adams, M. W. and Kelly, R. M. (1998) Finding and using    hyperthermophilic enzymes. Trends Biotechnol. 16, 329-332.-   C11. Li, W. F., Zhou, X. X. and Lu, P. (2005) Structural features of    thermozymes. Biotechnol. Adv. 23, 271-281.-   C12. Renugopalakrishnan, V., Garduno-Juarez, R., Narasimhan, G.,    Verma, C. S., Wei, X. and Li, P. (2005) Rational design of thermally    stable proteins: relevance to bionanotechnology. J. Nanosci.    Nanotechnol. 5, 1759-67.-   C13. Magliery, T. J. and Regan, L. (2004) Combinatorial approaches    to protein stability and structure. Eur. J. Biochem. 271, 1595-1608.-   C14. Eijsink, V. G., Gaseidnes, S., Borchert, T. V. and van den    Burg, B. (2005) Directed evolution of enzyme stability. Biomol. Eng.    22, 21-30.-   C15. Lehmann, M., Loch, C., Middendorf, A., Studer, D., Lassen, S.    F., Pasamontes, L., van Loon, A. P. and Wyss, M. (2002) The    consensus concept for thermostability engineering of proteins:    further proof of concept. Protein Eng. 15, 403-411.-   C16. Amin, N., Liu, A. D., Ramer, S., Aehle, W., Meijer, D., Metin,    M., Wong, S., Gualfetti, P. and Schellenberger, V. (2004)    Construction of stabilized proteins by combinatorial consensus    mutagenesis. Protein Eng. Des. Sel. 17, 787-793.-   C17. Polizzi, K. M., Chaparro-Riggers, J. F., Vazquez-Figueroa, E.    and Bommarius, A. S. (2006) Structure-guided consensus approach to    create a more thermostable penicillin G acylase. Biotechnol. J. 1,    531-536.-   C18. Bommarius, A. S., Broering, J. M., Chaparro-Riggers, J. F. and    Polizzi, K. M. (2006) High-throughput screening for enhanced protein    stability. Curr. Opin. Biotechnol. 17, 606-610.-   C19. Schlieker, C., Bukau, B. and Mogk, A. (2002) Prevention and    reversion of protein aggregation by molecular chaperones in the E.    coli cytosol: implications for their applicability in    biotechnology. J. Biotechnol. 96, 13-21.-   C20. Baneyx, F. and Mujacic, M. (2004) Recombinant protein folding    and misfolding in Escherichia coli. Nat. Biotechnol. 22, 1399-1408.-   C21. Kohda, K., Tsuji, Y., Takagi, M. and Imanaka, T. (1996) Cloning    and functional-analysis of molecular chaperone genes from Bacillus    stearothermophilus Sic1. Biotechnol. Lett. 18, 1061-1066.-   C22. Muchowski, P. J. and Clark, J. I. (1998) ATP-enhanced molecular    chaperone functions of the small heat shock protein human    αB-crystallin. Proc. Natl. Acad. Sci. USA 95, 1004-1009.-   C23. Yutani, K., Ogasahara, K., Tsujita, T. and Sugino, Y. (1987)    Dependence of conformational stability on hydrophobicity of the    amino acid residue in a series of variant proteins substituted at a    unique position of tryptophan synthase alpha subunit. Proc. Natl.    Acad. Sci. USA 84, 4441-4444.-   C24. Millard, C. B., Shnyrov, V. L., Newstead, S., Shin, I., Roth,    E., Silman, I. and Weiner, L. (2003) Stabilization of a metastable    state of Torpedo californica acetylcholinesterase by chemical    chaperones. Protein Sci. 12, 2337-2347.-   C25. González, M., Bagatolli, L., Echabe, I., Arrondo, J., Argarana,    C., Cantor, C. and Fidelio, G. (1997) Interaction of biotin with    streptavidin. J. Biol. Chem. 272, 11288-11294.-   C26. González, M., Argaraña, C. and Fidelio, G. (1999) Extremely    high thermal stability of streptavidin and avidin upon biotin    binding. Biomol. Eng. 16, 67-72.-   C27. Celej, M., Montich, G. and Fidelio, G. (2003) Protein stability    induced by ligand binding correlates with changes in protein    flexibility. Protein Sci. 12, 1496-1506.-   C28. Miyazaki, K., Wintrode, P. L., Grayling, R. A., Rubingh, D. N.    and Arnold, F. H. (2000) Directed evolution study of temperature    adaptation in a psychrophilic enzyme. J. Mol. Biol. 297, 1015-1026.-   C29. Polizzi, K. M., Bommarius, A. S., Broering, J. M. and    Chaparro-Riggers, J. F. (2007) Stability of biocatalysts. Curr.    Opin. Chem. Biol. 11, 220-225.-   C30. Villalonga, R., Tachibana, S., Cao, R., Ramirez, H. L. and    Asano, Y. (2006) Supramolecular-mediated thermostabilization of    phenylalanine dehydrogenase modified with beta-cyclodextrin    derivatives. Biochem. Eng. J. 30, 26-32.-   C31. Villalonga, R., Tachibanab, S., Caoc, R., Matosa, M. and    Asanob, Y. (2007) Glycosidation of phenylalanine dehydrogenase with    O-carboxymethyl-poly-beta-cyclodextrin. Enzyme Microb. Technol. 40,    471-475.-   C32. Abian, O., Grazu, V., Hermoso, J., Gonzalez, R., Garcia, J. L.,    Fernandez-Lafuente, R. and Guisan, J. M. (2004) Stabilization of    penicillin G acylase from Escherichia coli: site-directed    mutagenesis of the protein surface to increase multipoint covalent    attachment. Appl. Environ. Microbiol. 70, 1249-1251.-   C33. Iwai, H. and Plückthun, A. (1999) Circular beta-lactamase:    stability enhancement by cyclizing the backbone. FEBS Lett. 459,    166-172.-   C34. Scott, C. P., Abel-Santos, E., Wall, M., Wahnon, D. C. and    Benkovic, S. J. (1999) Production of cyclic peptides and proteins in    vivo. Proc. Natl. Acad. Sci. USA. 96, 13638-13643.-   C35. Giver, L., Gershenson, A., Freskgard, P. O. and    Arnold F. H. (1998) Directed evolution of a thermostable esterase    Proc. Natl. Acad. Sci. USA. 95, 12809-12813.-   C36. Zhang, N. Y., Suen, W. C., Windsor, W., Xiao, L., Madison, V.    and Zaks, A. (2003) Improving tolerance of Candida antarctica lipase    B towards irreversible thermal inactivation through directed    evolution. Protein Eng. 16, 599-605.-   C37. Hamamatsu, N., Nomiya, Y., Aita, T., Nakajima, M., Husimi, Y.    and Shibanaka Y. (2006) Directed evolution by accumulating tailored    mutations: thermostabilization of lactate oxidase with less    trade-off with catalytic activity. Protein Eng. Des. Sel. 19,    483-489.-   C38. Johannes, T. W., Woodyer, R. D. and Zhao, H. (2005) Directed    evolution of a thermostable phosphite dehydrogenase for NAD(P)H    regeneration. Appl. Environ. Microbiol. 71, 5728-5734.-   C39. Jones, S., Stewart, M., Michie, A., Swindells, M. B.,    Orengo, C. and Thornton, J. M. (1998) Domain assignment for protein    structures using a consensus approach: characterization and    analysis. Protein Sci. 7, 233-242.-   C40. Lehmann, M., Kostrewa, D., Wyss, M., Brugger, R., D'Arcy, A.,    Pasamontes, L. and van Loon, A. P. (2000) From DNA sequences to    improved functionality: using protein sequence comparisons to    rapidly design a thermostable consensus phytase. Protein Eng. 13,    49-57.-   C41. Lehmann, M., Wyss, M. (2001) Engineering proteins for    thermostability: the use of sequence alignments versus rational    design and directed evolution. Curr. Opin. Biotechnol. 12, 371-375.-   C42. Danson, M. J. and Hough, D. W. (1998) Structure, function and    stability of enzymes from the Archaea. Trends Microbiol. 6, 307-314.-   C43. Landenstein, R. and Antranikian, G. (1998) Proteins from    hyperthermophiles: stability and enzymatic catalysis close to the    boiling point of water. Adv. Biochem. Eng. Biotechnol. 61, 37-85.-   C44. Vieille, C. and Zeikus, G. J. (2001) Hyperthermophilic enzymes:    sources, uses, and molecular mechanisms for thermostability.    Microbiol. Mol. Biol. Rev. 65, 1-43-   45. Doi, N. and Yanagawa, H. (1999) Insertional gene fusion    technology. FEBS Lett. 457, 1-4.-   C46. Ostermeier, M. (2005) Engineering allosteric protein switches    by domain insertion. Protein Eng. Des. Sel. 18, 359-364.-   C47. Guntas, G. and Ostermeier, M. (2004) Creation of an allosteric    enzyme by domain insertion. J. Mol. Biol. 336, 263-273.-   C48. Evdokimov, A. G., Anderson, D. E., Routzahn, K. M. and    Waugh, D. S. (2001) Structural basis for oligosaccharide recognition    by Pyrococcus furiosus maltodextrin-binding protein. J. Mol. Biol.    305, 891-904.-   C49. Quiocho, F. A., Spurlino, J. C. and Rodseth, L. E. (1997)    Extensive features of tight oligosaccharide binding revealed in    high-resolution structures of the maltodextrin    transport/chemosensory receptor. Structure 5, 997-1015.-   C50. Hofnung, M., Bedouelle, H., Boulain, J. C., Clement, J. M.,    Charbit, A., Duplay, P., Gehring, K., Martineau, P., Saurin, W. and    Szmelcman, S. (1988) Genetic approaches to the study and use of    proteins: random point mutations and random linker insertions.    Bulletin de l'Institut Pasteur, 86, 95-101.-   C51. Betton, J. M., Jacob, J. P., Hofnung, M. and    Broome-Smith, J. K. (1997) Creating a bifunctional protein by    insertion of beta-lactamase into the maltodextrin-binding protein.    Nat. Biotechnol. 15, 1276-1279.-   C52. Betton, J. M., Martineau, P., Saurin, W. and Hofnung, M. (1993)    Location of tolerated insertions/deletions in the structure of the    maltose binding protein. FEBS Lett. 325, 34-38.-   C53. Guntas, G., Mansell, T. J., Kim, J. R. and    Ostermeier, M. (2005) Directed evolution of protein switches and    their application to the creation of ligand-binding proteins. Proc.    Natl. Acad. Sci. USA 102, 11224-11229.-   C54. Martineau, P., Leclerc, C. and Hofnung, M. (1996) Modulating    the immunological properties of a linear B-cell epitope by insertion    into permissive sites of the MalE protein. Mol. Immunol. 33,    1345-1358.-   C55. Puziss, J. W., Harvey, R. J., Bassford, P. J. Jr. (1992)    Alterations in the hydrophilic segment of the maltose-binding    protein (MBP) signal peptide that affect either export or    translation of MBP. J. Bacteriol. 174, 6488-6497.-   C56. Liang, J., Kim, J. R., Boock, J. T., Mansell, T. J. and    Ostermeier, M. (2007) Ligand binding and allostery can emerge    simultaneously. Protein Sci. 16, 929-937.-   C57. Hecky, J. and Muller, K. M. (2005) Structural perturbation and    compensation by directed evolution at physiological temperature    leads to thermostabilization of beta-lactamase. Biochemistry 44,    12640-12654.-   C58. Miller, G. L. (1959) Use of dinitrosalicylic acid reagent for    determination of reducing sugars. Anal. Chem. 31, 426-428.-   C59. Berrondo, M., Ostermeier, M. & Gray, J. J. (2008) Structure    prediction of domain insertion proteins from structures of    individual domains. Structure 16, 513-527.-   C60. Jung, W. S., Hong, C. K., Lee, S., Kim, C. S., Kim, S. J.,    Kim, S. I. & Rhee, S. (2007) Structural and functional insights into    intramolecular fructosyl transfer by inulin fructotransferase. J.    Biol. Chem. 282, 8414-8423.-   C61. Kim, K.-Y., Koo, B.-S., Jo, D. & Kim. S.-I. (2004) Cloning,    expression, and purification of exoinulinase from Bacillus sp.    Snu7. J. Microbiol. Biotechnol. 14, 344-349.-   C62. Kim, J. R. & Ostermeier, M. (2006) Modulation of effector    affinity by hinge region mutations also modulates switching activity    in an engineered allosteric TEM1 beta-lactamase switch. Arch.    Biochem. Biophys. 446, 44-51.-   C63. Gill, S. C. & von Hippel, P. H. (1989) Calculation of protein    extinction coefficients from amino acid sequence data. Anal.    Biochem. 182, 319-326.-   C64. Bradford, M. M. (1976) A Rapid and Sensitive Method for the    Quantitation of Microgram Quantities of Protein Utilizing the    Principle of Protein-Dye Binding. Anal. Biochem. 72, 248-254.-   C65. Melius, P. (1971) Isolation of yeast invertase by sephadex gel    chromatography. A biochemistry laboratory experiment. J. Chem. Edu.    48, 765-766.-   C66. McManus-Munoz, S. & Crowder, M. W. (1999) Kinetic mechanism of    metallo-beta-lactamase L1 from Stenotrophomonas maltophilia.    Biochemistry 38, 1547-1553.-   C67. Sigal, I. S., DeGrado, W. F., Thomas, B. J. & Petteway, S. R.,    Jr. (1984) Purification and properties of thiol beta-lactamase. A    mutant of pBR322 beta-lactamase in which the active site serine has    been replaced with cysteine. J. Biol. Chem. 259, 5327-5332.-   C68. Kumar, S., Adediran, S. A., Nukaga, M. & Pratt R F. (2004)    Kinetics of turnover of cefotaxime by the Enterobacter cloacae P99    and GCl beta-lactamases: two free enzyme forms of the P99    beta-lactamase detected by a combination of pre- and post-steady    state kinetics. Biochemistry 43, 2664-2072.-   C69. Sowek, J. A., Singer, S. B., Ohringer, S., Malley, M. F.,    Dougherty, T. J., Gougoutas, J. Z. & Bush, K. (1991) Substitution of    lysine at position 104 or 240 of TEM-1pTZ18R beta lactamase enhances    the effect of serine-164 substitution on hydrolysis or affinity for    cephalosporins and the monobactam aztreonam. Biochemistry 30,    3179-3188.

1. A method for protein stabilization by domain insertion into athermophilic protein.
 2. The method as claimed in claim 1, wherein theprocess is carried out without any modification of the primary sequenceof the protein and the intrinsic properties of activity and specificityof the protein are largely maintained.
 3. The method as claimed in claim2, wherein the domain insertion is accomplished by end-to-end fusion oftwo distinct proteins in which the N-terminus of one of the proteins isconnected to the C-terminus of the other of the proteins.
 4. The methodas claimed in claim 2, wherein the domain insertion is accomplished byinsertional fusion of two distinct proteins in a first of the proteinsis inserted into the middle of a second of the proteins.
 5. The methodas claimed in claim 4, wherein the insertional fusion is achieved insite-specific ways.
 6. The method as claimed in claim 4, wherein theinsertional fusion is achieved in random ways.
 7. The method as claimedin claim 4, wherein the insertional fusion involves recombinant DNAtechniques.
 8. The method as claimed in claim 4, wherein the thatinsertional fusion is carried out following an inactivation mechanism.9. The method as claimed in claim 8, wherein the inactivation mechanismis denaturation.
 10. The method as claimed in claim 4, wherein the firstprotein is wild-type exoinulinase (EI) and the second protein isPyrococcus furiosus (PfMPB).
 11. The method as claimed in claim 10,wherein the EI is inserted between residues 125 and 126 of the PfMBP.12. A method for protein stabilization by domain insertion into athermophilic protein, comprising inserting a first protein into a secondprotein using a fusion technique selected from the group consisting ofend-to-end fusion and insertional fusion, wherein the process is carriedout without any modification of the primary sequence of the protein, andwherein the intrinsic properties of activity and specificity of theprotein are largely maintained.
 13. The method as claimed in claim 12,wherein the domain insertion is accomplished by end-to-end fusion of twodistinct proteins in which the N-terminus of one of the proteins isconnected to the C-terminus of the other of the proteins.
 14. The methodas claimed in claim 12, wherein the domain insertion is accomplished byinsertional fusion of two distinct proteins in a first of the proteinsis inserted into the middle of a second of the proteins.
 15. The methodas claimed in claim 14, wherein the insertional fusion is achieved insite-specific ways.
 16. The method as claimed in claim 14, wherein theinsertional fusion is achieved in random ways.
 17. The method as claimedin claim 14, wherein the insertional fusion involves recombinant DNAtechniques.
 18. The method as claimed in claim 17, wherein the thatinsertional fusion is carried out following an inactivation mechanism.19. The method as claimed in claim 18, wherein the inactivationmechanism is denaturation.
 20. A method for protein stabilization bydomain insertion into a thermophilic protein, comprising insertingwild-type exoinulinase (EI) between residues 125 and 126 of Pyrococcusfuriosus (PfMPB) using insertional fusion, wherein: the process iscarried out without any modification of the primary sequence of theprotein; the intrinsic properties of activity and specificity of theprotein are largely maintained; the insertional fusion involvesrecombinant DNA techniques; and the insertional fusion is carried outfollowing an inactivation mechanism.